Screening And Functional Studies Of Transcriptional Factors And Coregulators Involved In Cellulase Gene Regulation In Trichoderma Reesei

Lignocellulose is one of the most abundant carbohydrates on earth.The degradation of this polysaccharides thus constitutes a major transformation step in the biological carbon cycle in nature.Filamentous fungi are the mostdominant microorganisms capable of efficiently degrading cellulose to glucose or cellulooligosaccharides in nature.Among those numerous filamentous fungus,Trichoderma reesei is one ofthe most prolific cellulase producers and is able to express and secrete large amounts of cellulases when exposed to cellulose.Thus T.reesei is now becoming one of the most important industrial strains to produce cellulases and hemicellulases.Despite the tremendous progress that has been made in the extensive characterization and physicochemical properties study of cellulase in T.reesei,the molecular mechanism underlying the regulation of cellulase production in this fungi is still insufficiently understood.At present,several transcriptional factors that are involved in regulatingcellulase gene expression have been identified in T.reesei,which containsfourtranscriptional activators(Xyrl,Ace2,Ace3 and Hap2/3/5)and two transcriptional repressors(Crel and Acel).Studies found that each transcriptioal factor has specific target nucleotide sequences on cellulase gene promoters,thus cellulase gene transcription in T.reesei is a complex process needing multiple transcriptional factors.So first of our work is screening and identification potential new transcriptional factors.In eukaryotic cells,nucleosome and chromatin structure play critical roles intranscriptional regulation.Two classes of chromatin remodeling enzymes have beenshown to facilitate transcription of chromatin templates in vivo,which including the histone acetyltransferases and the ATP-dependent remodelingenzymes.So we also focus on the mechanism that how the chromatin remodeling complex SWI/SNF complex regulate the induced expression of cellulase genes in T.reesei.The main results of this study are as follows:1.Rce1 was identified as a noveltranscriptional repressor regulates cellulase gene expression by antagonizing the transactivator Xyrl in T.reeseiRce1 was identified as a novel transcriptional repressor using cbh1 as a bait in yeast-one hybrid.Rce1 was a nuclear protein and was annotated as a fungal-specific transcriptional factor due to the presence of a typical zinc binuclearcluster domain.Disruption of the rce41 gene facilitated the induced expression of cellulasegenes while overexpression of rce1 significantly reduced the expression of cellulase genes,which indicated that Rce1 was a transcription repressor of cellulase genes.Electrophoretic mobility shift(EMSA)and DNase Ifootprinting assays demonstrated that Rce1 could bind directly to a cbh1 gene promoter region containing a clusterof Xyrl binding sites.Furthermore,competitive binding and ChIP assays revealed that Rce1 antagonized Xyrl frombinding to the cbhl promoter in regulation of cellulase expression at initial induction stage.Thesimultaneous overexpression of Xyr1 in the OErce1 strain(OExyr1?OErcel)fully restored cellulase gene expression as evidenced by the almost normal extracellular cellulase production.In conclusion,Rce1 reduced cellulase gene expression through antagonized Xyr1 from binding to the cellulase gene promotes.2.TrUbc4 plays an important role in regulation cellulase gene expressionTrUbc4 was identified as a ubiquitin conjugating enzyme using cbhl as a bait in yeast-one hybrid.TrUbc4 was a nuclear protein and belong to UBC family.Disruption of the Trubc4 gene reduced the induced cellulase expression whilecomplementation of the ?Trubc4 strain with the Trubc4gene expressed under native promoter from its endogenouslocus restored the normal induction kinetics,and disruption of other two ubiquitin conjugating enzymegenes had hardly effect on cellulase expression,thus indicated that TrUbc4 was a specific ubiquitin conjugating enzyme that response to the cellulose induction signal.Mutation analysis revealed that the active site Cyc8 5 of TrUbc4 played a vital role in cellulase expression.Overexpression ofxyrl in ?Trubc4strain could not restore the induced expression level of cellulases genes on cellulose.ChIP analyses showed that deletion of Trubc4 reduced the occupation of Xyrl on cellulase gene promoters both in the wild type or OExyr1backgrounds.TrSnf12 was identified as an interacting protein of TrUbc4 in yeast two hybrid.TrSnf12 containing a SWIB/MDM domain is also a nuclear protein and disruption of its encoding gene showed the similar phenotype withTrubc4.Regrettably,we failed to detect the Ub-ligase activity of TrSnfl2 in vitro self-ubiquitination assays with some uncertain factors.Taken together,these results indicated that ubiquitin conjugating enzyme TrUbc4 played an important role in cellulase expression.3.Throughing ubiquitome analysis found TrUbc4 may play roles in many physiological process in T.reesei.In this ubiquitome work with TU6 and ?Trubc4strains,2563 ubiquitination sites and 1385 proteins were identified,and 1923 ubiquitination sites and 941 protein were quantified.Among the quantified ubiquitination sites,396 were up-regulated and 298 were down-regulated over 1.5 folds.Interesting,there were 295 ubiquitination sites and 258 proteins were indentified in TU6 but not in ?Trubc4.Theses proteins refer to transcriptional factors,ubiquitin ligase,kinases,transportors and SAGA comples,which implying that TrUbc4 may play roles in many physiological process in T.reesei.4.Xyr1 Recruits SWI/SNF to cellulase gene promoters in regulation cellulase gene expressionTrSnf12 interacted with the activation domain(AD)of Xyr1 in yeast two hybrid and GST pull down assay.Furthermore,TrSnfl2 also interacted with the core subunitTrSwi1 in yeast two hybrid.So we speculate that Xyr1 had an interaction with Swil through TrSnf12.On the basis of the previous studies,we found that deletion Trsnfl2 reduced the cellulase expression.Similarly,we also analyzed the two other core subunits of SWI/SNF complex Trswil/Trsnf5in cellulase expression.We found that disruption ofTrswil and Trsnf5 abolished the cellulase expression and transcription.And this two knockout strains also showed a growth defect on solid plate and conidiation,but had no effect on growth in liquid medium with glucose as a carbon source.ChIP analyses revealed that Swi1 and TrSnf5 was recruited to cellulase gene promoters under cellulose inducting conditions.Further analysis revealed that the TrSwi1 was also recruited to cellulase gene promoters under non-inducing conditions when xyr1 was overexpressed while was not in wild type.These results thus indicated that Swi1 was recruited to cellulase gene promoters in a Xyr1 dependent manner and this recruitment was mediated by the interactions between TrSnf12 and Xyr1.However,overexpression of xyr1 in ?Trswi1 or ?Trsnf5 could mainly bypass the effect of deletion phenotype.Taken together,Xyr1 Recruits SWI/SNF to cellulase gene promoters in regulation cellulase expression through interaction with TrSnf12.